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Investigating the E. Coli DNA Polymerase V Mutasome
by
Meghna Patel
———————————
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
MOLECULAR BIOLOGY
May 2013
Copyright 2013 Meghna Patel

In Escherichia Coli, cell survival and genomic stability after UV radiation or other DNA damaging agents depends on repair mechanisms induced as part of the SOS response. The SOS response involves upregulation of over 40 genes, which are normally repressed by the LexA protein under non-damage conditions. The early phase of the SOS response is centered around error-free DNA repair mechanisms. These include base excision repair (BER), nucleotide excision repair (NER), and recombinational repair. If these mechanisms are insufficient to surmount the replicational impedance brought about by DNA damage, the bacterial cell advances into the later part of the SOS response. This later phase of the response helps ensure cell survival albeit at the cost of faithful DNA repair. Known as SOS mutagenesis, this phase is characterized by elevated mutation levels, which are a direct result of error-prone DNA replication. SOS mutagenesis is the result of the low fidelity DNA polymerase V. DNA polymerase V (Pol V) is a heterotrimeric protein composed of the catalytically active UmuC and a dimer of UmuD'. The heterotrimer is essentially inactive, however, and must strip a RecA monomer and an ATP cofactor from the 3' tip of a RecA-DNA nucleoprotein filament to form an active Pol V mutasome composed of UmuC:UmuD'₂:RecA:ATP. This dissertation focuses on investigating the Pol V mutasome including deducing protein-protein interactions in the active, inactive, and de-activated states of the mutasome, detecting a conformational change in the different activity states of the mutasome, visualizing the SOS response in live cells, and attempting to ascertain the crystal structure of Pol V.

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1
Investigating the E. Coli DNA Polymerase V Mutasome
by
Meghna Patel
———————————
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
MOLECULAR BIOLOGY
May 2013
Copyright 2013 Meghna Patel